Cellulose is one of the major components in plant cell wall and is also a major resource of biomass on earth. Hence, many enzymes that degrade cellulose can be widely applied in many different industries. Cellulose is a polysaccharide composed of glucose units linked by β-1,4-glycosidic bond. These polysaccharides organize tightly together to form crystalline cellulose in order to defense destructing energy from outside of plant. On the other hand, many kinds of herbivores and microbes need to degrade cellulose from plant to glucose as an energy source by different degrading enzymes including cellulase, xylanase and so on. The catalytic mechanism of cellulase involves hydrolyzing the β-1,4-glycosidic bond between two sugar units by acid-base interaction. Cellulase can be generally divided into three groups including endoglucanase, cellobiohydrolase and β-glucosidase. Endoglucanase can randomly degrade cellulose into many small fragments. Cellobiohydrolase can degrade cellulose from reducing end or non-reducing end to release main product, cellobiose. β-Glucosidase can degrade cellobiose into simple sugar glucose.
So far, the industrial applications of cellulase are widespread in food industry, feed industry, textile industry or paper pulp industry, even in biofuel production. In general, cellulase needs to conform to different appropriate conditions according to different industrial needs. For example, acidic and thermostable enzymes are suitable for the feed industry but textile industry prefers alkaline enzymes. Therefore, scientists always try to seek better enzymes which are more suitable for different industrial needs in academic or industrial researches. Currently, many researchers and enzyme companies could produce better enzymes by screening in nature or modifying present enzymes. There are generally two strategies of enzyme modification including directed evolution that randomly mutates the enzyme gene and selects with desirable properties or rationale engineering that specifically mutates the enzyme gene based on the structural information of the enzyme.
Different industrial production processes need different appropriate enzymes to cooperate and participate in their production procedures. Despite cellulase has been applied in industry for a long time, many industrial cellulases which are produced from mesophile such as Trichoderma reesei have worse thermostabilities. On the other hand, thermostable cellulase can be efficiently applied in the industry which needs high temperature reaction condition, such as brewing, bioethanol production and so on. Thermostable enzyme has higher protein stability, so it can be stable and even work better in high temperature condition. In addition, to increase enzyme activity is also a key point for the improvement of industrial enzyme. Higher enzyme activity represents the cost down and the companies will have better profit.
According to previous studies, disulfide bonds are beneficial to protein stability and thermostability. Trichoderma reesei has many kinds of cellulases, in which the cellulase Cel5A belonging to GH family 5 and whose protein structure (ID 3QR3) had been published in 2011 has four disulfide bonds at positions C16-C22, C92-C99, C232-C2683 and C273-C323, and thus has high melting temperature (Tm). Cel5A belongs to α/β TIM-barrel protein (Toni M Lee, Mary F Farrow, Frances H Arnold, and Stephen L Mayo. (2011) Protein Structure Report, November27; 20(11):1935-40). In 2004, Simon R. Andrews et al. found that adding disulfide bonds at N terminals and C terminals of the xylanase CjXyn10A of Cellvibrio japonicas and the xylanase CmXyn10B of Cellvibrio mixtus had increased the protein stability and further increased the protein thermostability, and both CjXyn10A and CmXyn10B belong to α/β TIM-barrel proteins (Andrews S. R., Taylor E. J., Pell G., Vincent F., Ducros V. M., Davies G. J., Lakey J. H., and Gilbert H. J., (2004) J. Biol. Chem. December 24;279(52):54369-79).
Therefore, the present invention intends to add disulfide bonds of a cellulase by gene modification, so as to increase the thermostability and further increase the industrial value of the cellulase.